Abstract:

The present invention provides a CTLA-4 non-blocking agent of a complete
human antibody nature, thus is non-immunogenic in a human. The
immunoassay method using such a non-blocking agent measures the CTLA-4
content in a sample of a human subject. The present invention further
provides a novel method for isolating human regulatory T cells. The
resultant enriched and depleted cellular populations are useful in
treating or ameliorating of human diseases.

Claims:

1. A composition for detecting human CTLA-4, said composition
comprising:(a) a CTLA-4 antagonist or agonist, wherein said antagonist
comprises an antibody or an antigen-binding fragment thereof and said
agonist comprises CD80 or an CTLA-4-binding fragment thereof; and(b) a
CTLA-4 non-blocking agent that recognizes the extracellular domain of
CTLA-4, wherein said CTLA-4 non-blocking agent comprises an antibody or
an antigen-binding fragment thereof.

2. The non-blocking agent or functional derivative of claim 1 that binds
to the sequences consisting SEQ ID NO:1.

3. The non-blocking agent of claim 1, wherein the agent is a human
monoclonal antibody recognizes human CTLA-4 with a Kd of about 4 nM or
less.

4. The non-blocking agent of claim 1, wherein the agent is a human
IgG4.lamda. antibody.

5. A method of isolating regulatory T cells from human samples, said
method comprising:(a) contacting a population of suspended cells in a
sample with a CTLA-4 non-blocking agent that recognizes the extracellular
domain of CTLA-4, wherein said CTLA-4 non-blocking agent is
non-immunogenic in a human and comprises an antibody or an
antigen-binding fragment thereof; and(b) selecting cells that bind to the
CTLA-4 non-blocking agent, wherein the selected cells are enriched for
regulatory T cells.

6. The non-blocking agent or functional derivative of claim 5 that binds
to the sequences consisting SEQ ID NO:1.

7. The non-blocking agent of claim 5, wherein the agent is a human
monoclonal antibody recognizes human CTLA-4 with a Kd of about 4 nM or
less.

8. The non-blocking agent of claim 5, wherein the agent is a human
IgG4.lamda. antibody.

9. The isolated population of regulatory T cells of claim 5, wherein the
cells also express FOXP3.

10. The method of claim 5 further comprising transfusing the enriched
population of said regulatory T cells into a subject to suppress the
autoimmune response.

11. The method of claim 10 wherein the isolated and enriched cellular
population is expanded in cell culture before transfusing into a subject.

12. The method of claim 5 wherein the exclusive cellular populations of
step (b) that do not bind to the CTLA-4 non-blocking agent are deficient
in regulatory T cells.

13. The method of claim 12 further comprising introducing said depleted
cellular population into a subject to enhance the activation of T cells.

14. The method of claim 13 wherein the depleted cellular population
further contacts an antigen in cell culture before introducing into a
subject.

15. A method for increasing the activation of T cells in a human subject
comprising administrating an effective amount of a functional derivative
of the CTLA-4 non-blocking agent, wherein the functional derivative of
the CTLA-4 non-blocking agent binds to the sequences consisting SEQ ID
NO:1.

Description:

FIELD OF THE INVENTION

[0001]This invention relates to the study of human CTLA-4 (cytotoxic T
lymphocyte antigen-4, or CD152) that represents an essential receptor
involved in negative regulation of T cell activation. More particularly,
it relates to the identification of an antigen specific to human CTLA-4
molecule, a complete human monoclonal antibody which specifically binds
to the antigen, and various uses for the monoclonal antibody, such as
detection and isolation agents.

[0054]All of the publications, patents and patent applications cited above
or elsewhere in this application are herein incorporated by reference in
their entirety to the same extent as if the disclosure of each individual
publication, patent application or patent was specifically and
individually indicated to be incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0055]On-going studies of the collaboration scenarios between T cells and
antigen-presenting cells (APCs) foresaw dramatic changes which have
revealed some therapeutic targets and medicinal potentials in certain
regards. For example, it has been shown that the specialized and dynamic
molecular machinery, present in the tight junction between a T cell and
an APC, regulates immunological responses (Dustin, 2002; Grakoui et al.,
1999; Qi et al., 2001). It has also been inferred that the machinery,
termed the immunological synapse, correlates with a high degree of
intercellular communication controlling disparate biological process
(Davis and Dustin, 2004). A number of molecules have been confined at the
immunological synapse to ensure their interim expression and interaction
at the right time and place thus the sum and integration of signals are
relevant to evoke appropriate T cell responses. Within this limited and
m-sized area is full of interacting molecules, of which CTLA-4 has been
identified to be responsible for inhibiting T cell responses in a T cell
receptor (TCR)-dependent manner (Chikuma and Bluestone, 2002; Egen and
Allison, 2002).

[0056]Human CTLA-4 was mapped to band q33 of chromosome 2 and was
classified into a group of immunomodulating receptors, collectively
termed as CD28 superfamily (Sharpe and Freeman, 2002). As shown in FIG.
1, the complete cDNA sequence of human CTLA-4 (isoform A) has the Genbank
accession number L15006 and the structure has the accession number 1AH1
in the Molecular Modeling DataBase (MMDB) of NCBI's structure database.
The region of amino acids -35 to 0 is the singal peptide; 1-126 is the
extracellular V-like domain; 123-151 is the transmembrane domain; and
152-188 is the cytoplasmic domain. It is well established that two
members of this superfamily, CD28 and CTLA-4, have opposing functions and
that CTLA-4 represents one of the major inhibitory receptors involved in
co-stimulatory pathways regulating both humoral and cellular immune
responses (Krummel and Allison, 1996; Linsley et al., 1991; Pioli et al.,
2000). A majority of studies indicate that CD28 provides direct
enhancement signals, including up-regulation/stabilization of cytokine
gene transcription, improved cell survival, lowered threshold for
activation, and cytoskeletal effects; however, information on the
function of CTLA-4 is much less clear. Thus far, the most compelling
evidence for an inhibitory role of CTLA-4 is derived from the deficient
knockout mice (CTLA-4-/-) (Tivol et al., 1995). These mice suffer
from a fatal T-cell lymphoproliferative disorder with splenomegaly,
lymphoadenopathy and hyper-responsive infiltration in several organs
including heart that become apparent by four weeks after birth. This
fetal disorder is presumably due to reactivities to multiple
self-antigens, since the expression of a single transgenic TCR prevents
this disease. The TCR-dependent activation in these knockout mice appears
to require CD28 costimulation, because CTLA-4-/-CD28-/- mice do
not suffer the lymphoproliferative disease. Likewise, treatment of these
mice perinatally with soluble CTLA-4-Ig, which competes ligands access of
cell surface CTLA-4, prevents such a disease effectively. Nonetheless,
the mechanism of CTLA-4 action is still unclear, with no obvious central
theme.

[0057]Conceptually, the interaction of CD28 on the lymphocyte with B7
proteins on the APC provides a necessary costimulatory second signal for
a T cell to be able to fully respond to an antigen. The original family
members in the pathway consist of two B7 ligands--CD80 (B7-1) and CD86
(B7-2), which have specificities towards the two receptors--CD28 and
CTLA-4. CD28 is constitutively expressed on the surface of T cells
whereas CTLA-4 surface expression is rapidly up-regulated to a limited
extent following T cell activation. The kinetics of expression of CD80
and CD86 also differ. CD86 is constitutively expressed on interdigitating
dendritic cells, Langerhans cells, peripheral blood dendritic cells,
memory B cells and germinal center B cells. Furthermore, CD86 is
expressed at low levels on monocytes, but its rapid up-regulation through
IFN-γ stimulation has led to the hypothesis that CD86 functions
primarily in initiating an immune response. On the other hand, CD80,
being expressed later in time, may serve to amplify or regulate the
response. Newly identified family members of the related molecules
include: the inducible costimulatory molecule (ICOS), program death 1
(PD-1) receptor, B and T lymphocyte attenuator (BTLA), B7-H1, B7-H2,
B7-H3, B7-H4, PD-1 ligand 1 (PD-L1) and PD-L2 (Wang and Chen, 2004). The
novel interactions among these new family members underscore additional
complexity of this costimulatory pathway in mounting an appropriate
immune response.

[0058]A shorter soluble form of CTLA-4 lacking the transmembrane region
has been achieved from RT-PCR cloning of non-activated T cells in animals
as well as humans (Magistrelli et al., 1999; Oaks et al., 2000). Soluble
CTLA-4 (sCTLA-4, CTLA-4 isoform B; FIG. 1) seems to be a fully functional
CD80 and CD86 receptor, thus likely to affect T-cell responses in a
paracrine manner. Furthermore, immunoreactive sCTLA-4 can be detected in
the serum of 14/64 healthy subjects. In addition, the presence of high
concentration of sCTLA-4 was observed in sera of patients with autoimmune
thyroid diseases such as Graves' disease (Oaks and Hallett, 2000).
Finally, recent reports show that sCTLA-4 levels are augmented in
patients with autoimmune diseases, such as type-1 diabetes (Nistico et
al., 1996), diffuse cutaneous systemic sclerosis (Sato et al., 2004),
systemic lupus erythematosus (Wong et al. (2005) Rheumatology 44:989),
and allergic asthma (Wong et al. (2005) Clin. Exp. Immunol. 141:122). It
has been shown that activated T cells suppress sCTLA-4 mRNA expression
and express preferentially the membrane-bound, full-length CTLA-4
(flCTLA-4) mRNA (Gough et al., 2005). Thus the ratio of sCTLA-4 to
flCTLA-4 may have an important role in the regulation of immune
homeostasis. The alternate transcripts or spliced variants of sCTLA-4,
which lack the transmembrane encoding regions, were first deposited in
the GenBank Sequence Database in humans, mice, and rats (accession
numbers U90273, U90270, and U90271) in 1997 followed by a description of
the same transcript in humans, being expressed by non-stimulated human T
cells. The endogenous 174-aa soluble form, designed as isoform B, can be
retrieved under the accession number NP--001032720.

[0059]It is also known in the field that immune reactivity is further
controlled by various types of regulatory T cells (Tregs) (Sakaguchi,
2005). Tregs can be broadly divided into two subsets, i.e., the natural
Treg cells of CD4.sup.+ CD25.sup.+ phenotype, which constitute 5-10% of
peripheral T cells, and the stimulation-induced (or adaptive) Treg cells
identified in various models of inflammation, alloreactivity, or
autoimmunity (Prud'homme, 2004). Recent findings suggest that the
suppressive potential of CD4.sup.+ CD25.sup.+ natural Tregs to other
activated effector T cells is mediated by restricting early proliferation
and the anti-effector function in inflamed tissues (von Boehmer, 2005).
The forkhead-family transcription factor gene FOXP3, encoding the scurfin
transcriptional regulator (Genbank accession number EF534714, NCBI
protein accession number ABQ15210), has been implicated in the
development and function of natural Tregs (Hori et al., 2003). A FOXP3
mutation in scurfy mice results in the absence of Tregs and early death
from a multi-organ inflammatory disorder similar to the CTLA-4 or
TGF-β deficiency. FOXP3 was shown to function as a transcriptional
repressor, targeting composite NF-AT/AP-1 sites in cytokine gene
promoters and the region responsible for NF-AT inhibition was mapped to
the amino terminus (Lopes et al., 2006; Wu et al., 2006).

[0060]In principle, conventional techniques to isolate this rare Treg
population often involve a two-step, multiple antibody selection
procedure (Miltenyi Biotec, Bergisch Gladbach, Germany; BD Biosciences
Pharmingen, San Jose, Calif.). Briefly, CD4.sup.+ T lymphocytes are first
preserved from not binding to a cocktail of mAbs that recognize other CD
antigens expressed on erythrocytes, platelets, monocytes and peripheral
leukocytes, etc. Subsequently, anti-human CD25 mAb positively selects the
CD25.sup.+ cells from the enriched CD4.sup.+ cells, yielding CD4.sup.+
CD25.sup.+ Treg cells. However, inevitably intermittent exposure to
environmental pathogens results in traditional effector T cell activation
and consequently expression of CD25 on human CD4.sup.+ T cells, making
identification of the Treg population a very difficult task.
Additionally, even CD4-CD8.sup.+ natural Treg cells have been
reported by Xystrakis et al. (2004) Blood 104: 3294-3301, indicating that
Treg is a heterogeneous population. Furthermore, although FOXP3
expression is found predominantly within the Tregs, its intracellular
nuclear localization causes direct detection impossible to live cells.
Therefore, the characterization and application of Treg cells have been
hampered by a lack of specific molecular markers on the surface of Tregs.
A more complex approach was engineered to circumvent this particular
problem, in which purified CD4.sup.+ CD25.sup.+ peripheral blood
mononuclear cells are further activated with agents such as ionomycin,
and the Tregs are isolated based on binding to CTLA-4 blocking mAb
(Birebent et al., 2004). Yet an even more complicated process has evolved
by using additional surface markers like CD45RA and CD127 (WO
2007/117602).

[0061]Of interest is the association and potential synergism between the
suppressive function of Tregs and the CTLA-4 expression. Unusually for
non-activated T cells, Tregs constitutively express CTLA-4 (Takahashi et
al., 2000), and CTLA-4 blockade on the Treg by specific blocking mAb can
attenuate their suppressive activity, leading to the development of
autoimmune disease in vivo (Read et al., 2006). In addition, it has been
observed not only that the reported CD4-CD8.sup.+ natural Tregs
express CTLA-4 (Xystrakis et al. 2004) but also that CD4.sup.+ CD25.sup.+
cells further purified on the basis of recycling CTLA-4 are much more
potent as regarding suppression (Birebent, et al., 2004). More
importantly, the fact that inducible Tregs were the dominant source of
sCTLA-4 was revealed in the 2007 British Society for Immunology Congress
(Ward and Barker, 2007). Together, they indicate a strong correlation
between CTLA-4 expression and suppressive regulatory function, supportive
of the concept that CTLA-4 is functionally relevant to Tregs.

[0062]Because Tregs, in accompany with sCTLA-4, are involved in preventing
allograft rejection and graft versus host disease and exert a dominant
effect in controlling autoimmunity and maintaining peripheral tolerance,
specific immune therapies designed to isolate and then expand them may
improve the clinical course of various T-cell mediated pathology. As
CTLA-4 provides the most important attenuating costimulatory signals, it
will be expected by one of skill in the art that these molecules offer
new targets for immunotherapy and diagnostics.

[0063]Studies of the physiological function and practical uses of CTLA-4
became possible with the isolation of monoclonal antibodies (mAbs). The
first reported mouse anti-human CTLA-4 mAb (clone 11D4) suggested that
blocking CTLA-4 signaling might deliver a positive signal synergizes with
that delivered by CD28 (Linsley et al., 1992). The immune-enhancing
nature of CTLA-4 antagonism has thus opened the possibility for a readily
applicable tumor immunotherapy by temporary removal of CTLA-4-mediated
inhibition using antagonistic Abs (Egen et al., 2002). Although the
mechanisms by which CTLA-4 regulates T cell responses are not completely
understood, blocking its activity with an antagonistic or blocking mAb
offers a novel approach that holds a promise for immunotherapy. A set of
corresponding U.S. patents such as U.S. Pat. No. 5,811,097, U.S. Pat. No.
5,855,887, U.S. Pat. No. 6,051,227, U.S. Pat. No. 6,207,156 and U.S. Pat.
No. 7,229,628, illustrates approach of CTLA-4 blockade to strongly
enhance antitumor responses has been highly regarded for the treatment
potentials.

The anti-CTLA4 blocking mAbs, e.g., clone BNI3 (Steiner et al., 1999)
(commercially available from BD Pharmingen) and clone AS33 (Antibody
Solutions, Mountain View, Calif.), are often in use to detect sCTLA-4 in
biological fluid (Oaks and Hallett, 2000) and to purify Tregs from
activated peripheral blood (Birebent, et al., 2004). However, these may
not be the best available strategy. Structural analyses have shown that
the human CTLA-4 protein is composed of disulfide-linked homodimers of
extracellular immunoglobulin variable (IgV) domains, each domain
consisting of two layered β-sheets with ten strands (A, A', B, C,
C', C'', D, E, F and G) that form three complementarity determining
region (CDR)-like regions (Schwartz et al., 2001; Stamper et al., 2001).
Together with one mutational study (Peach et al., 1994), these two
structural studies have independently pointed out that CDR1-like (the B-C
loop) and CDR3-like (the F-G loop) regions in CTLA-4 directly bind
endogenous B7 ligands (CD80 and CD86), whereas CDR2's responsibility is
very trivial if there is any. Therefore, although in the initial
publications there is no definite information to describe the CTLA-4
epitope on which the blocking mAbs bind, antagonistic effects and the
subsequent enhancement on T-cell activation may be mediated by mAb
competition that results from specific binding with amino acid residues
on or close to a room encompassing CDR1 and CDR3. Thus uses of blocking
mAbs in pair or in combination with endogenous B7 ligands provide a
possible limitation caused by steric hindrances.

SUMMARY OF THE INVENTION

[0064]In one aspect, the present invention pertains to the discovery
herein that potions other than CDR1 and CDR3 of human CTLA-4, such as the
Met 55-cored CDR2-like sequence, do not play a role in binding of B7
ligands. Accordingly, mAbs that recognize a pre-determined Met 55-cored
region can be used and are particularly useful to detect sCTLA-4 and/or
to purify Tregs. To explore the effect of this Met 55-cored CDR2-like
sequence, the inventors have recently developed and had possession of a
complete human monoclonal IgG4λ targeting this particular stretch
(Chin et al., 2007). As the mAb is capable, under the condition that the
binding of a natural agonist was not interrupted, to mediate high
(nanomolar) affinity binding to an extracellular constituency
encompassing the CDR2-like region of CTLA-4, whereby it can react to
activated human CD3.sup.+ cells, thus the invention can further provide a
method for detecting sCTLA-4 and/or isolation Tregs.

[0065]The method contemplated herein may lead to an increase in
sensitivity for sCTLA-4 detection and thus may be used to diagnose those
conditions in which disease activity is tightly associated with sCTLA-4
production. Assays of interest include ELISA, RIA, FIA and flow
cytometry, etc. In one embodiment, the agent is selected from the group
consisting of: an antibody to CDR2 region of CTLA-4, a blocking antibody
to CTLA-4, or a preferred combination of an antibody to CDR2 region of
CTLA-4 and labeled human CD80. Binding may be quantified by a variety of
methods known in the art. After an incubation period sufficient to allow
the binding to reach equilibrium, the insoluble support is washed, and
the remaining label quantified. The preferred agents in combination will
enhance the detected label in the presence of sCTLA-4 and thus increase
the detection limit.

[0066]On the other hand, the present invention may lead to an increase in
efficiency for Treg isolation and encompasses both in vitro and in vivo
methods. For in vitro uses, the cell intrinsically possessing the CTLA-4
receptor without prior activation, i.e., Tregs, may be purified from
peripheral blood mononuclear cells. Efficient purification strategies are
known in the art, including depletion and enrichment. Strategies of
interest include magnetic cell separation, panning, bead-based
chromatography and cytometry sorting, etc. As an example, purified mAb
can be bound to an insoluble support, e.g. microtiter plate, magnetic
beads, etc. The candidate cells are added to the support, and the unbound
components are then washed off. The target cells are finally purified or
isolated by elution. As to in vivo methods, the Treg may be present in a
mammal, especially a human subject such as one who is suffering from
declined or excessive Treg levels and who could benefit from a respective
increase or decrease in Treg cells. Potential patients include those who
have low or no level of Treg and develop autoimmune diseases that require
Treg transfusion (Rao et al., 2007; Wildin and Freitas, 2005), or those
who undergone adoptive immunotherapy that demand Treg reduction (Dannull
et al., 2005). Thus, the invention provides a method for repopulating
Treg cells in a human comprising administering to the human a
therapeutically effective amount of a mAb.

[0067]In an embodiment, the invention provides antibodies that
specifically bind to the Met 55-cored CDR2-like region of human CTLA-4.
Preferred antibodies are monoclonal antibodies (mAbs) which are
non-immunogenic in a human and bind to an epitope in the extracellular
domain of CTLA-4. A preferred form of mAbs is human monoclonal
IgG4λ, or a fragment thereof. More preferably, suitable mAbs will
have an equilibrium dissociation constant (Kd) at least about 10-6 M
toward human CTLA-4, more preferably at least about 10-8M. The
antibody is preferably an IgG antibody, particularly IgG4.

[0068]According to a further aspect, the invention is concerned with the
CTLA-4 and a soluble form of this particular receptor which is the CTLA-4
extracellular domain. The mAbs against the Met 55-cored CDR2-like region
are optionally conjugated with, or fused to, molecules which increase the
serum half-lives thereof and can be formulated as pharmaceutical
compositions comprising the mAbs and a physiologically acceptable
carrier. Antibodies which bind to the Met 55-cored CDR2-like region may
optionally be fused to a heterologous polypeptide or magnetic particles
and the antibody or fusion thereof may be used to isolate and purify
CTLA-4.

[0069]In further embodiments, antibodies which bind to CTLA-4 may
optionally be fused or linked to a toxin and the antibody or fusion
thereof may be used to separate or kill Treg cells from a source of human
lymphocytes. Methods to fuse or link are known in the art, including
genetic and chemical techniques. Genetic manipulations may include
constructing an artificial nucleic acid segment consists of amino acid
residues of a toxin molecule and antigen-binding domains derived from a
mAb and producing said construct in a proper host, as described in WO
2005/012495. The immunotoxin may also be obtained from chemical
conjugations such as using a heterobifunctional reagent (e.g.,
N-succinimidyl 3-(2-pyridyldithio)propionate), carbodiimide linkage or
mixed anhydride procedure (Burstein and Knapp, 1977). The toxin moiety
can be, e.g., any of the following toxic polypeptides: ricin, pseudomonas
exotoxin, bryodin, diphtheria toxin, gelonin, α-sarcin,
aspergillin, restrictocin, angiogenin, saporin, abrin, pokeweed antiviral
protein, or a functional fragment of any of these toxic polypeptides.

[0072]FIG. 3 demonstrates the immunological and biochemical natures of the
anti-CTLA-4 mAb. Panel A shows isotyping and subtyping results by
immobilizing anti-human Igs as indicated. The binding profile of the mAb
was subsequently revealed by biotinylated CTLA-4-muIg and
avidin-peroxidase conjugates. Results indicate that the present mAb
belongs to a type of IgG4λ. Panel B indicates the isoelectric point
of the monoclonal IgG4λ anti-CTLA-4 (lane 4 and 8), resolved by
isoelectric focusing. Monoclonal human myeloma IgG1λ (lane 2 and 6)
and IgG4λ (lane 3 and 7) were run in parallel for comparison. The
electrophoretic patterns were visualized by either Coomassie brilliant
blue staining (lane 1-5) or immunoblot (lane 6-8) with anti-human IgG
conjugated with peroxidase and FAST® DAB (Sigma). The calculated
isoelectric points for human IgG1λ, IgG4λ and anti-CTLA-4 mAb
to be approximately in the range of 7.92-8.79, 5.76-6.52 and 7.87-8.41,
respectively, based on the calibration against the linear regression of
standard protein markers. Panel C outlines the affinity determination by
IAsys. Surface plasmon resonance obtained at 25° C. for increasing
concentrations of anti-CTLA-4 mAb on purified, unlabeled CTLA-4-muIg. The
straight line in the inset was obtained from the kobs plot versus
ligated Ab concentration and yielded a kdiss (the intercept) of
16.81 and a kass value (the gradient) of 4.20×109.
Therefore produced a Kd (kdiss/kass) of 4×10-9 M.

[0073]FIG. 4 indicates the binding of the present anti-CTLA-4 and
CD80/CD86 agonists to human CTLA-4 are not mutually exclusive. Results
obtained from ligand competition assays that test the ability of the
complete human monoclonal anti-CTLA-4 (solid line) and BNI3 (dashed line)
anti-CTLA-4 to compete for the CD80/CD86 and CTLA-4 interactions.
Biotinylated CD80-muIg or CD86-muIg plus indicated increasing
concentrations of the mAbs (10-3-10 μg/mL) were incubated in
microtiter wells coated with purified CTLA-4-muIg. Bound CD80/CD86 was
detected with avidin-peroxidase conjugate and a peroxidase substrate. The
data shown are representative of three experiments.

[0074]FIG. 5 is a representative combined data from the ELISA on human
CTLA-4 detection, using available mAbs and recombinant human CD80 to
CTLA-4. The assay was measured using commercially available CTLA4-muIg as
a standard. ELISA analysis showing the limit of sensitivity of 0.39, 1.56
and 6.25 ng/ml for the assay using a pair of the human monoclonal
IgG4λ and CD80 (.diamond-solid.), the human IgG4λ and BNI3
(.tangle-solidup.) and blocking mAbs of BNI3 and AS-33 (•),
respectively.

[0075]FIGS. 6A and 6B present the effect of isolating Tregs based on
intrinsic CTLA-4 surface expression. In FIG. 6A, detection of FOXP3 gene
expression relative to GAPDH expression by real-time PCR analysis in
purified CD4.sup.+ CD25- (.box-solid.), CD4.sup.+ CD25.sup.+
(.tangle-solidup.) and CTLA-4.sup.+ () T cells from three normal donors
is shown. Shown in FIG. 6B, pronounced enhancements, as compared with the
control group (.box-solid.), in the kinetics of thymidine incorporation
were observed when Tregs were removed by the uses of CTLA-4.sup.+ () or
anti-CTLA-4-diphtheria toxin conjugate (.tangle-solidup.). It was
difficult to consistently recover CD4-CD25- population and thus
omitted from the analysis. The proliferation response of CD4.sup.+
CD25.sup.+ (◯) and CTLA-4.sup.+ cells (∇) is
indicated. The data represent the mean of triplicate samples.

DETAILED DESCRIPTION OF THE INVENTION

[0076]In describing the present invention, the following terms will be
employed, and are intended to be defined as indicated below.

DEFINITIONS

[0077]A "complete human antibody" is an antibody containing exclusively
human sequences. The antibody is preferably a monoclonal antibody. The
terms "CTLA-4" when used herein encompass the native human sequence of
CTLA-4 isoform A (FIG. 1). Optionally, the CTLA-4 is not associated with
native glycosylation. "Native glycosylation" refers to the carbohydrate
moieties which are covalently attached to CTLA-4 when it is produced in
the mammalian cell from which it is derived in nature. Accordingly, human
CTLA-4 produced in a non-human cell is an example of a CTLA-4 which is
"not associated with native glycosylation". Sometimes, CTLA-4 is
unglycosylated as a result of being produced recombinantly in a
prokaryote or being synthesized chemically.

[0078]"Soluble CTLA-4" or "sCTLA-4" is a CTLA-4 molecule which contains
neither a transmembrane domain nor a cytoplasmic tail and represents the
native human sequence of CTLA-4 isoform B (FIG. 1). The "CTLA-4
extracellular domain" is a form of CTLA-4 which is essentially free of
the transmembrane and cytoplasmic domains of CTLA-4. Ordinarily, the
"CTLA-4 extracellular domain" will have an amino acid sequence of least
about 95% amino acid sequence identity with the amino acid sequence of
CTLA-4 isoform B indicated in FIG. 1, preferably includes CDR1-, CDR2-
and CDR3-like regions.

[0079]An "antigenic function" means possession of an epitope or antigenic
site that is capable of cross-reacting with antibodies raised against a
native sequence of CTLA-4 defined by the CDR-like regions. The principal
antigenic function of a CDR2-like region is that it does not involve in
binding of CTLA-4, sCTLA-4 or CTLA-4 extracellular domain to B7
molecules.

[0080]"Immunize" a cell or an animal with an antigen refers to the action
of exposing the cell or the animal to the antigen. The cell or animal can
be immunized in any manner that leads to contact between the cell or the
animal with the antigen.

[0081]A "heteromyeloma cell line" is a cell line derived from fusion of
two different myeloma cells. The two different myeloma cells are
preferably a human myeloma cell and a murine myeloma cell. Heteromyeloma
cell lines are known in the art. For example, U.S. Pat. No. 6,228,361 and
Chin et al., 2001 describe the preparation, characterization and use of
various heteromyeloma cell lines.

[0082]A "fusion partner" is a cell that can be used to fuse with an
antibody-producing cell for a beneficial purpose. Typically, the fusion
leads to prolonged antibody production. Thus, without fusion to the
fusion partner, the antibody-producing cell ceases to produce antibodies
in culture. Upon fusion to the fusion partner, however, fused cells can
be selected that produce antibodies in culture for at least about 3
months, preferably at least about 6, 9, 12, 18, 24 months or more. Fusion
partners include, but are not limited to, myeloma cells and heteromyeloma
cells.

[0083]An "agonist" is a molecule that can bind to cellular receptors,
e.g., CTLA-4, and thus can produce various biological effects and
initiate changes in cell function. Endogenous agonists are generally
nature-occurring ligands such as neurotransmitters and, in the case of
CTLA-4, CD80 and CD86. Exogenous agonists are commonly found as drugs.

[0084]An "antagonist" is a molecule that can bind to receptors, e.g.,
CTLA-4, but do not activate signal transduction mechanisms. The
biological effects of a given antagonist are derived from preventing
agonist binding and receptor activation, e.g., blocking mAbs.

[0085]"Non-immunogenic in a human" means that upon contacting the
polypeptide of interest in a physiologically acceptable carrier and in a
therapeutically effective amount with the appropriate tissue of a human,
no state of sensitivity or resistance to the polypeptide of interest is
demonstrable upon the second administration of the polypeptide of
interest after an appropriate latent period e.g., 8 to 14 days. It will
be understood by one of skill in the art that a polypeptide of complete
human origin typically represents "non-immunogenic in a human".

[0086]"Treating or ameliorating" a disease or medical condition means
reducing or eliminating the symptoms of the disease or medical condition,
or slowing down the progress of the disease/medical condition. The
reduction is preferably at least about 10%, more preferably at least
about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0087]An "effective amount" is an amount of an agent that is sufficient to
result in the intended effect. For example, for an antibody used to treat
or ameliorate a disease, an effective amount is an amount of the antibody
sufficient to reduce or eliminate the symptoms of the disease, or to slow
down the progress of the disease.

[0088]A "sample" is an aliquot or a representative portion of a substance,
material, or population. For example, a sample may be a sample of water,
sewage, oil, sand, blood, biological tissue, urine or feces. A
"biological sample" is a sample collected from or present within a
biological subject, preferably a human subject.

[0089]"Essentially pure" protein means a composition comprising at least
about 90% by weight of the protein, based on total weight of the
composition, preferably at least about 95% by weight. "Essentially
homogeneous" protein means a composition comprising at least about 99% by
weight of protein, based on total weight of the composition.

[0090]As used herein, the term "antibody" is used in the broadest sense
and specifically covers monoclonal antibodies, antibody compositions with
polyepitopic specificity, bispecific antibodies, diabodies, and
single-chain molecules, as well as antibody fragments (e.g., Fab,
F(ab')2, and Fv), so long as they exhibit the desired biological
activity.

[0091]The term "monoclonal antibody (mAb)" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the population are
identical except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies are highly specific,
being directed against a single antigenic site (epitope). The modifier
"monoclonal" indicates the character of the antibody as being obtained
from a substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any particular
method.

[0092]A "functional derivative" of a mAb is a compound having a
qualitative biological property in common with a native mAb protein.
"Functional derivatives" include, but are not limited to, fragments of
native sequenced mAb, provided that they have a biological activity in
common with a corresponding native sequenced mAb. The phrase "fragment"
as used in connection with mAb fragmented derivatives, such as
immunotoxins, provided that they have a biological activity in common
with a corresponding native sequenced mAb.

[0093]A "magnetic cell separation" or "magnetic flow sorting" is a
technique of cell selection based on achieving steady-state isolation
between magnetic and non-magnetic cell streams in a flowing suspension.
The selectivity depends upon cell tagging with cell surface marker
antibodies labeled with a magnetic colloid. The characteristic feature of
the method is its capability to fractionate cells based on the surface
antigen expression. In order to perform this technique for the purpose of
positive selection or depletion of cells labeled with human IgG4λ
mAb (as described in Example 3), 1×108 single-cell suspension
of PBMC can be first labeled with the mAb at 20 μg/ml reaction buffer
for 15 min at 4° C. After unbound mAb is removed by washing, 0.2
ml of mouse anti-Human IgG MicroBeads (Miltenyi Biotec) is added and
develops 15 min at 4° C. Following washing steps to remove unbound
MicroBeads, the resuspended cells are magnetically separated in a
magnetic field provided by the manufacturer (VarioMACS Separator). This
isolated cell population represents cells intrinsically express CTLA-4 on
their surface, and thus stands for Tregs.

[0094]A "thymidine incorporation assay", which evaluates the capability of
cells to expand, can be used to measure the suppression of T cell
proliferation in response to a recall antigen by isolated Tregs. In order
to perform this assay, 5×105/well PMBC from vaccinated donors
are activated with 5 μg/ml of tetanus toxoid (TT) as described in
Example 3. During TT-driven proliferation, the cells are plated out in
96-well culture dishes containing a test sample with or without isolated
Tregs (such test samples are optionally diluted) and cultured for three
to seven days in a cell culture incubator at 37° C. in 5% CO2
and air. Proliferation was measured by 3H-thymidine incorporation.
During the final 16 hours of an assay, 1 μCi of 3H-thymidine is
added to each well for the last 16 and proliferation is measured by
scintillation counting using a Packard TopCount® Microplate
Scintillation Counter (PerkinElmer, Shelton, Conn.). Tregs are expected
to induce a statistically significant decrease (to a P value of 0.05) in
3H-thymidine uptake, relative to control. Preferred Treg cells lead
to a decrease in 3H-thymidine uptake which is at least 30% of that
of the control.

Modes and Methods for Carrying Out the Invention

[0095]The present invention is based on the discovery of the human
monoclonal IgG4λ mAbs against the Met 55-cored CDR2-like region of
CTLA-4. The epitope thus defined is shared by both CTLA-4 and sCTLA-4.
The experiments described herein demonstrate that this mAb is a complete
human mAb which appears not to play a role in the binding of CTLA-4 to
its endogenous B7 ligands. In particular, this antibody has been found to
enhance CTLA-4-CD80 interaction in certain conditions, thus indicating
that it may be used to detect sCTLA-4 in combination with human CD80. As
the presence of nature ligands of CTLA-4 is common in preparation of
peripheral blood mononuclear cells (PBMC), other uses for this mAb, e.g.
single-step enrichment of human Tregs, will be apparent and evident from
the following discussion. A description follows as to how such a mAb may
be prepared.

[0096]Culture materials and reagents are known in the art and may be
obtained commercially. The culture medium used is RPMI-1640 (HyClone,
Logan, Utah), supplemented with 1× non-essential amino acids (Life
Technologies, Gaithersburg, Md.), 10% fetal bovine serum (FBS; Life
Technologies) and 50 μg/ml of gentamycin and kanamycin (Sinton
Chemical & Pharmaceutical, Hsinchu, Taiwan). Purified and biotinylated
human CTLA-4-murine Ig fusion protein (rhCTLA-4-murine Ig or CD152-muIg),
CD80-muIg and CD86-muIg (Ancell, Bayport, Minn.) are used in
antigen-specific and competing enzyme-linked immunosorbent assay (ELISA),
together with peroxidase-labeled goat antibodies against human IgG and
IgM (Zymed Laboratories, South San Francisco, Calif.) or avidin
horseradish peroxidase (eBioscience, San Diego, Calif.) as the reporting
system. The fluorochrome-conjugated mouse mAb against human IgGs and
human CD3 (UCHT1; mouse IgG1), together with rat mAb against mouse IgG2a
are commercially available from Becton Dickinson Immunocytometry Systems
(San Jose, Calif.) and Abcam (Cambridge, UK). The anti-CD3 (OKT3; mouse
IgG2a) uses for T cell activation and the antagonistic anti-CD152 (BNI3;
mouse IgG2a) can be purchased from eBioscience and Abcam, respectively.

[0097]Complete human mAbs are produced from in vitro stimulation and
culture techniques. Generally, plasma and buffy coat samples from healthy
routine blood donors, screened negative for HIV-1/2, HTLV-I/II, HCV,
HBsAg and containing normal levels of alanine transferase (ALT), can be
obtained from local Blood Centers. PBMC are isolated by density
centrifugation on Ficoll-Paque (GE Healthcare Bio-Sciences, Uppsala,
Sweden) as described elsewhere. The resulting PBMC are magnetically
labeled with CD45RO MACS® microbeads (Miltenyi) then separated by a
VarioMACS® (Miltenyi) instrument according to the manufacturer's
instructions. The purified CD45RO.sup.+ T cells are cultured at a density
of 2×106 cells/ml in the culture medium supplemented with 50
μM 2-mercaptoethanol and 10 μg/ml pokeweed mitogen (PWM; Sigma, St.
Louis, Mo.). After 24 h, cells are removed by 400×g centrifugation
to collect CD45RO.sup.+ T cell replacing factor. Removal of cytotoxic
cell populations is similarly performed by using colloidal
super-paramagnetic microbeads conjugated to monoclonal anti-human CD8 and
anti-CD56 antibodies (Miltenyi). Removal of IL-10-producing cells may be
achieved by using rat anti-human IL-10 (SouthernBiotech, Birmingham,
Ala.) and goat anti-rat IgG microbeads (Miltenyi).

[0098]Site-directed in vitro immunization is preformed by using cytotoxic
cell-depleted PBMC based on a two-step principle. Primary immunization is
performed by incubating the cells for 6 days in a medium containing 10 nM
of the heterotopic peptide antigen (QYIKANSKFIGITELAATYMMGNELTFLDDSICT;
Fine Research Biochem, Taoyuan, Taiwan), 50 μM 2-mercaptoethanol, 10%
heat-inactivated human serum, 0.05 ng/ml recombinant human (rh) IL-2
(eBioscience), and 25% (v/v) CD45RO.sup.+ T cell replacing factor. For
secondary immunization, 3×107 primary-immunized cells are
mixed with the peptide in a flask that had been immobilized overnight
with 5 mg/ml of CD40L (CD154; eBioscience) together with 1×107
QYIKANSKFIGITEL (Fine Research Biochem)-stimulated CD4.sup.+ T cells and
5 ng/ml rh IL-15 (eBioscience). The cells are cultured for 3-5 days in a
medium supplemented with 5% human serum, 50 mM 2-mercaptoethanol and 10
nM heterotopic peptide antigen. The significance of differences between
treated and control cultures can be established by a variety of
statistical methods known in the art such as Student's t test.

[0101]Instead of fusion, the in vitro immunized cells can be used to
construct an antibody library, and the antibodies of interest are then
identified from this library. Thus, after in vitro immunization,
antibody-producing cells can be identified with the antigen (the cells at
this stage can be optionally infected with EBV). A phage-display library
is then constructed using these antibody-producing cells, and the phages
containing the antibody fragment of interest can be identified by
screening this library with the antigen. The methods of constructing
phage display libraries are known in the art (Duenas et al., 1996).

[0102]To define the specific epitope of human CTLA-4 recognized by the
mAb, peptide arrays (Genesis Biotech, Taipei, Taiwan and Fine Research
Biochem, Taoyuan, Taiwan) containing in-situ synthesized peptides
immobilized on special membrane can be used. In brief, 1 μg/mL of
protein A (Proteus MIDI kit, Pro-Chem, Littleton, Mass.)-purified mAb is
incubated by shaking in room temperature for 2 h. After washing, the
membrane-bound mAb can be then visualized by diluted anti-human IgG
conjugated with peroxidase (Jackson ImmunoResearch Laboratories, West
Grove, Pa.) and FAST DAB (Sigma). The amount of bound mAb is calculated
by Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, Md.) on
the scanned images.

[0103]Assays to determine affinity and specificity of binding are known in
the art, including competitive and non-competitive assays. A
non-competitive assay is preferred in this analysis. The affinity of the
mAb can be determined against rhCTLA-4-murine Ig fusion protein with an
IAsys® optical biosensor (Affinity Sensors, Cambridge, UK) according
to the manufacturer's instructions. Briefly, 200 μg/ml dialyzed and
diluted rhCTLA-4-murine Ig is immobilized on the activated surface of
carboxymethyl dextran cuvettes in 10 mM of sodium acetate buffer at pH
3.8. After conditioning with 10 mM HCl, immobilization of 2 mg/mL
CD152-muIg can result in a response of 1100 arc sec. This represents the
highest immobilization response for CD152 and gives a ligate binding
capacity (Rmax) of 300 arc sec. Serial dilutions of the mAb in PBS,
i.e. 1.34×10-9 M, 6.70×10-9 M, 1.34×10-8
M, 2.68×10-8 M and 5.36×10-8 M, are added to the
CD152-coated cuvettes (final volume, 50 μl). Affinity constants (Kd)
are calculated from these measurements as kdiss/kass by using
the FASTFIT® program provided by the manufacturer.

[0104]The present invention provides a novel method of measuring human
sCTLA-4 in biological samples. To this end, we develop an immunoassay for
quantification sCTLA-4, and show in Example 2 that a soluble form of
CTLA-4 can be detected at least ten times of lower sensitivity as
compared with a conventional method known in the art. For illustration,
sandwich ELISAs are used for detection of sCTLA-4 in human serum. For
this purpose, wells of a 96-well microtiter plate were coated with
anti-CTLA4 blocking mAb (clone BNI3; BD Pharmingen) or non-blocking
CTLA-4 mAb as the insoluble support. After saturation by bovine serum
albumin, 1001 of a 1:3 dilution of the test samples are applied to the
wells, and the plates are incubated for 60 min at room temperature and
then wash to remove unbound material. Next, a reporting system containing
either biotinylated anti-CTLA4 mAb (clone AS-33, Antibody Solutions) or
biotinylated CD80-muIg (Ancell) is added, and the reactions are further
incubated for 1 h. Reactions are developed using a streptavidinperoxidase
complex (Zymed) and 3,39,5,59-tetramethyl-benzidine substrate. Optical
density (OD) is read at 450 nm. A standard curve can be generated with
the use of a dilution series of a commercially available CTLA4-Ig fusion
protein (Ancell). Binding may be measured by a variety of methods known
in the art. After an incubation period sufficient to allow the binding to
reach equilibrium, the insoluble support is washed, and the remaining
label quantified. Assays of interest include ELISA, RIA, FIA and flow
cytometry, etc.

[0105]Also provided is a method of isolating Treg cells from a sample,
comprising contacting a suitable sample with the antibody, or functional
derivatives thereof, of the present invention so as to form an
antibody-antigen complex between the antibody and any CTLA-4 present on
the surface of cells in the sample, and detecting the presence of any
complex so formed, thereby isolating in the sample the presence of
extracellular CTLA-4. It is intended that the isolation of Tregs can be
performed by using only one positive selection step, i.e., preserving
cells specifically express extracellular CTLA-4. Therefore, both
populations of Treg and non-Treg can be recovered with minimal in vitro
manipulation and maximal viability. To this end, in Example 3,
1×108 single-cell suspension of PBMC can be first labeled with
the mAb at 20 μg/ml reaction buffer for 15 min at 4° C. After
unbound mAb is removed by washing, 0.2 ml of mouse anti-Human IgG
MicroBeads (Miltenyi Biotec) is added and develops 15 min at 4° C.
Following washing steps to remove unbound MicroBeads, the resuspended
cells are magnetically separated in a magnetic field. This isolated cell
population thus represents Treg cells intrinsically express CTLA-4 on
their surface. Consequently, negatively selected non-Treg population can
be subsequently stimulated, e.g., by a recall antigen, without prolonged
selection steps and thus without compromise of their survival.

[0106]Also in Example 3, a functional derivative, i.e., immunotoxin, is
prepared from conjugation of diphtheria toxin with purified IgG4λ.
Purified IgG4λ mAb is mixed with six times excess of N-succinimidyl
3-(2-pyridyldithio)propionate (GE Healthcare Bio-Sciences) in PBS, and
the mixture is allowed to react for 30 min at room temperature and then
dialyzed against PBS. The modified IgG4λ is then mixed with three
times excess of reduced diphtheria toxin (Merck Taiwan LTD., Taipei,
Taiwan) and 10-fold concentrated PBS (10% of the total volume) and store
for 36 h at 40° C. The product is dialyzed and concentrated with
Macrosep® Centrifugal Devices (Pall Corporation, East Hills, N.Y.)
equilibrated and washed with PBS.

[0107]Suitable samples which are useful in the methods of CTLA-4 detection
and Treg isolation include, but are not limited to biological fluids from
a human subject such as blood, nasal mucosal discharge, oral mucosal
discharge, vaginal mucosal discharge, semen, purrulent exudates, anal
mucosal discharge and synovial fluid. Samples may also include lymphoid
tissues such as spleen, lymph nodes, thymus, bone marrow, tonsils and
Peyer's patches. In one embodiment, the human antibody is labeled with an
immunological retrievable marker, i.e., anti-human Ig antibody conjugated
with magnetic beads. Such a specific binding may be retrieved by a
variety of methods known in the art, including but not limited to direct
labeling of the presented mAb, cell panning, and fluorescence-activated
cell sorting.

[0108]It has been generally accepted that human CD4.sup.+ CD25.sup.+ Treg
cells express FOXP3, whereas CD25-T cells do not and the expression
of FOXP3 in CD4.sup.+ T cells correlates with their ability to function
as Treg cells (Sakaguchi, 2005). In one embodiment, the expression of
FOXP3 is quantified by quantitative real-time PCR (QPCR) analysis in
which RNA is first extracted using an RNeasy Mini Kit (Qiagen, Valencia,
Calif.) according to the manufacturer's instructions, and cDNA is then
prepared with 2.5 μM random hexamers (Applied Biosystems Inc., Foster
City, Calif.). Message levels can be quantified by real-time PCR System.
Amplification is carried out in a total volume of 25 μl for 40 to 50
cycles of 15 seconds at 95° C., 1 minute at 60° C., and
product may be detected using SYBR Green I dye (Molecular Probes Inc.,
Eugene, Oreg.). Samples are run in triplicate, and their relative
expression was determined by normalizing expression of each target to
GAPDH, and then comparing this normalized value to the normalized
expression in a reference sample to calculate a fold-change value.
Primers were designed so that amplicons spanned intron/exon boundaries to
minimize amplification of genomic DNA. Primer sequences were as follows:

[0109]The antibody or its functional derivatives, e.g., immunotoxins, may
be administered by any suitable method known in the art, such as via
intravascular, intrathecal, intravenous, intramuscular, parenteral,
subcutaneous, intramedullar, intraperitoneal, topical, oral, rectal,
vaginal, nasal, pulmonary and intratumoral routes.

Compositions

[0110]Another aspect of the present invention provides a composition
comprising a fully human antibody prepared according to the present
invention. Preferably, the antibody binds to its antigen with a high
affinity. The Kd is preferably about 100 nM or less, more preferably
about 40 nM or less, yet more preferably about 10 nM or less, still more
preferably about 4 nM or less, and most preferably about 1 nM or less. In
particular, the antibody is capable of recognizing at least two related
antigens, such as microbial antigens that are only different due to
antigenic variation, or proteins encoded by alleles of the same gene.

[0111]This invention also includes pharmaceutical compositions that
contain, as the active ingredient, one or more of the antibodies in
combination with a pharmaceutically acceptable carrier or excipients. In
preparing the compositions of this invention, the active
ingredient/antibody is usually mixed with an excipient, diluted by an
excipient or enclosed within such a carrier which can be in the form of a
capsule, sachet, paper or other container. When the pharmaceutically
acceptable excipient serves as a diluent, it can be a solid, semi-solid,
or liquid material, which acts as a vehicle, carrier or medium for the
active ingredient. Thus, the compositions can be in the form of solutions
(particularly sterile injectable solutions), tablets, pills, powders,
lozenges, sachets, cachets, elixirs, suspensions, emulsions, syrups,
aerosols (as a solid or in a liquid medium), ointments containing, for
example, up to 10% by weight of the antibody, soft and hard gelatin
capsules, suppositories, and sterile packaged powders.

[0112]Some examples of suitable excipients include lactose, dextrose,
sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,
alginates, tragacanth, gelatin, calcium silicate, microcrystalline
cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and
methyl cellulose. The formulations can additionally include: lubricating
agents such as talc, magnesium stearate, and mineral oil; wetting agents;
emulsifying and suspending agents; preserving agents such as methyl- and
propylhydroxy-benzoates; sweetening agents; and flavoring agents. The
compositions of the invention can be formulated so as to provide quick,
sustained or delayed release of the active ingredient after
administration to the patient by employing procedures known in the art.

[0113]The liquid forms in which the novel compositions of the present
invention may be incorporated for administration by injection include
aqueous solutions (such PBS), suitably flavored syrups, aqueous or oil
suspensions, and flavored emulsions with edible oils such as corn oil,
cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as
elixirs and similar pharmaceutical vehicles.

[0114]The following examples are offered to illustrate this invention and
are not to be construed in any way as limiting the scope of the present
invention.

[0144]To characterize the nature of complete human mAb binding, epitope
mapping was performed by the Western blotting method with arrays
containing the overlapping pendecapeptides, encompassing CDR1-like (the
B-C loop), CDR3-like (the F-G loop) and the Met 55-cored sequence
localized between the C' and D strands of the CD152 extracellular
portion. FIG. 2 depicts that only the peptide corresponding to the
C-terminus of the Met 55-cored sequence (54 YMMGNELTFLDDSIC68;
SEQ ID NO:1) was best recognized by the mAb, and thus representing the
epitope, while neither the promiscuous T-cell epitope used to boost in
vitro stimulation, nor CDR1-like or CDR3-like region contributes to the
binding. From this result, it can be concluded that the Ala 51, Ala 52,
Thr 53 and Thr 69 are not essential for mAb recognition.

b) Immunological and Biochemical Natures of the mAb

[0145]The essentially pure mAb was isotyped and subtyped by solid phase
ELISA, utilizing its reactivity with human CTLA-4 and appropriate
immobilized typing Abs. The binding depicts the simultaneous presence of
γ4 and λ chains in the mAb while other Ig chains are absent
(FIG. 3A), thus the mAb has a γ4λ human Ig phenotype.
Additionally, to compare the clonal nature with existing human monoclonal
IgG1λ and IgG4λ derived from purified myeloma proteins, the
isoelectric focusing (IEF) patterns were subsequently visualized. The
resolvable bands, as shown in FIG. 3B, indicate that the presented mAb
has a slightly lower yet basic pI similar to the IgG1λ, but in
contrast to the acidic IgG4λ myeloma protein or to the anionic (pI
4.5-5.0) species of proteins commonly described for polyclonal IgG4.
Western blot confirmed the purity of the Ab samples as illustrated by the
anti-λ staining configurations. As shown in FIG. 3B, the
corresponding pI of the myeloid IgG1λ, IgG4λ and the
presented mAb obtained with linear regression of pH gradient were
7.92-8.79, 5.76-6.52 and 7.87-8.41, respectively. The equilibrium
dissociation constant (Kd) for the purified intact mAb was determined by
an IAsys analysis. The rate constant was evaluated directly from the
sensogram using five cycles of soluble mAb binding to the immobilized
rhCTLA-4-muIg. FIG. 3D reveals that, with the analysis of extent and
association in single phase, the Kd was deduced to be 4×10-9M.

c) Little or No Competition to B7-CD152 Binding of the mAb

[0146]Although the CDR2-containing epitope does not seem to be involved in
the binding of endogenous cognate ligands (CD80 and CD86), the epitope
might present an allosteric site for non-competitive inhibition. To
investigate this possibility, rhCTLA-4-muIg was immobilized onto wells
and either biotinylated CD80-muIg or CD86-muIg was used as a binding
ligand in the presence of the mAb or BNI3, i.e., a CTLA-4 blocking mouse
IgG2a mAb (Steiner, et al., 1999). FIG. 4 shows, in contrast with the
expected dose-dependent inhibition of specific receptor binding by the
antagonistic BNI3, the mAb could not compete binding significantly with
either CD80-muIg or CD86-muIg. Surprisingly, high doses of the mAb
display synergism with the natural ligand CD80 but not CD86 with a
consequence up to 50% enrichment of CD80 binding to rhCTLA-4-muIg.

Example 2

Using CDR2-Specific Agent Increase the Detection Limits of Human CTLA-4

[0147]In practice, immunoassays based on the use of pairs of blocking Abs,
which reactive with epitopes within the B7-binding region of the
molecule; confront a primary limitation of steric hindrance. Blocking Abs
may compete with each other and with endogenous CD80 and CD86 for the
restricted CDR1- and CDR3-like regions, making them difficult to
distinguish native sCTLA-4 in an assay system. To determine whether the
above CTLA-4 binding synergism with the natural ligand CD80 displayed by
the mAb may contribute a lower sensitivity; the mAb was coupled with CD80
in detecting sCTLA-4. In brief, the ELISA system utilizing the
IgG4λ-coated plate for capture and biotinylated CD80-muIg
recombinant protein (Ancell) for detection, followed by
streptavidin-peroxidase for color reaction. FIG. 5 shows a representative
experiment. This combination predominantly resolves a range of CTLA-4
from 0.39 to 50 ng/ml, while the blocking mAb pair identifies 6.25 to 50
ng/ml under similar experimental conditions. Likewise, a higher
resolution of 1.56 to 50 ng/ml was also observed when the present mAb was
in use together with a blocking mAb. This is not only consistent with the
predicted respective three-dimensional binding sites based on the
previous studies (Linsley, et al., 1992; Schwartz, et al., 2001; Stamper,
et al., 2001) but also demonstrative that CTLA-4 mAb with CDR2-like
region specificity, in combination with agents of CDR1- and CDR3-like
specificities, increases the detection limits of CTLA-4.

Example 3

Isolation of the Intrinsic CTLA-4.sup.+ Populations Resulted in Increased
Foxp3 Expression and Suppression

[0148]One of the major unresolved problems in the field of using Tregs as
a promising alternative to the standard immunosuppression regime is how
to develop a reliable method for their isolation. Most prior arts
reported to date exploit a negative selection non-CD4.sup.+ cells and a
positive selection CD25.sup.+ cells, which is time-consuming and
experience-dependent to manipulate human PBMC. However, having
acknowledged the importance of CTLA-4 in Treg's function (Read, et al.,
2006; Ward and Barker, 2007), the present invention turned the attention
to utilize intrinsic CTLA-4 surface expression for Treg isolation. In
particular, we asked if the non-blocking anti-CTLA-4 mAb provides a
single positive selection for Teg cells. To address this question, we
employed a thymidine incorporation assay to detect the suppression of
built-in Tregs and the enhancement of Treg removal.

[0149]By analyzing the subsets from the in vitro tetanus toxoid
stimulation for proliferation, removal of CTLA-4.sup.+ population is of
great benefit to a recall response (FIG. 6B), further arguing that this
is a Treg population. In some donors, the majority of CD4-CD25cells could be recovered from the bound magnetic beads, but not from
other donors. It is not known what causes the variability among donors.
The identity of the population that upregulated CTLA-4 surface expression
was also determined. The cells which upregulated CTLA-4 expression were
enriched for Treg cells, and this supported by the fact that these cells
retained antigen unresponsiveness similar to the purified CD4.sup.+
CD25.sup.+ population. Because CD4.sup.+ CD25.sup.+ cells predominantly
represent Tregs and CTLA-4 surface expression is associated with an even
higher FOXP3 expression, it was thus confirmed that CTLA-4.sup.+ cells
symbolize regulatory T cells.